SH 179 

j.T8 L6 

I /Copy 1 





BookJxl 



The Action of Various Pharmaco- 
logical and other Chemical 
Agents on the Chromatophores 
of the Brook Trout Salvelinus 
Fontinalis Mitchill 



JOHN N. f,OWE 

From the Department of Zoology, University of 
Wisconsin 



Reprinted from The Journal of Experimental 
ZooLOGT, Vol. 23, No. 1, May, 1917 

UNIVERSITY OF WISCONSIN 
PH. D. THESIS i<^ 1.^- 



SHl7f 

.T?Le 



I 



J Reprinted from The Journal of Experimental Zoology, Vol. 23, No. 1 

May, 1917 



<:''0 



THE ACTION OF VARIOUS PHARMACOLOGICAL AND 

OTHER CHEMICAL AGENTS ON THE CHROMA- 

TOPHORES OF THE BROOK TROUT SAL- 

VELINUS FONTINALIS MITCHILL 

JOHN N. LOWE 
From the Department of Zoology, University of Wisconsin 

THREE TEXT FIGURES AND ONE PLATE 

CONTENTS 

Material and methods 148 

Reactions to gases 150 

1. Oxygen 150 

2. Carbon dioxide 150 

Effect of distilled water 151 

Reactions to salts 152 

1. Effects of potassium salts 153 

2. Effects of sodium salts 154 

3. Discussion 156 

Reactions to alcohols 163 

1. Methyl alcohol 164 

2. Ethyl alcohol 164 

3. Propyl alcohol ; 167 

Reactions to alkaloids 169 

1. Strychnine 170 

2. Picrotoxin 172 

3. Morphine 173 

4. Caffeine 174 

5. Curara 176 

6. Nicotine ; 178 

7. Atropine. . •. 179 

8. Cocaine 180 

9. Veratrine ISl 

10. Quinine 182 

Summary 183 

Bibliography 187 

The reaction,s of melanophores (pigment cells) to pharmaco- 
logically active agents have been but little investigated. In the 

■ 147 



148 JOHN N. LOWE 

majority of the physiological researches upon the melanophores, 
the experiments have only included the study of such physical 
agents as light, heat, etc. The problem here undertaken was to 
determine the reactions of the melanophores of young trout em- 
bryos in response to changes in their chemical enviroimient. The 
trout embryos that were used in these experiments were too 
young to react to a change in the light conditions, and through- 
out the work gave no evidence of any psychic influence of the 
pigment cells. 

MATERIAL AND METHODS 

Young brook trout embryos, from two days to two weeks after 
hatching, were used. The melanophore of such young individuals 
are dark, much branched cells with deep black or brown pigment 
granules. These are the only kind of pigment cells present at 
this time. The xanthophores, the yellow or reddish pigment 
cells, appear after or a little before the yolk is absorbed. All the 
experiments were performed before the xanthophores appeared. 
After the yolk is absorbed the fish begin to react to the back 
ground. When placed in a da,rk dish, they become dark; when 
placed in a white dish, light in color. Microscopical examina- 
tion shows that the pigment cells (melanophores) are expanded 
in the dark colored individuals and contracted in the light ones. 
The very young, two-day or two-week old embryos do not 
respond to changes of the back ground. 

This constant condition is taken as a known factor. The 
contraction of the pigment cells was used as the criterion for 
determining stimulation, and their expansion (relaxation) as a 
mark of depression. The expansion of the pigment cells is char- 
acterized by the peripheral migration of the pigment granules 
within the processes of cell, and in contraction the movement is 
centripetal. My reason for considering contraction as stimula- 
tion and expansion as a depression is that certain reagents, alka- 
loids for example, if used in high concentrations produce no ob- 
servable change in the pigment cells which under normal con- 
ditions are expanded. Small or 'therapeutic' doses produced a 



CHEMICAL AGENTS ON CHROMATOPHORES 149 

contraction. Large doses produced an expansion of all the cells 
which had contracted in the weak solution. Inasmuch as it has 
been shown by various investigators that large doses of pharma- 
cologically active agents produce a depression, and small doses 
incite a stimulation in other tissues, it is inferred that the 
condition is essentially the same with the melanophores. 

All the chemicals used in these experiments were of Merck's, 
Kahlbaum's and Baker's manufacture. The solutions were 
made up with oxygenated distilled water. Chemically pure 
oxygen was bubbled through the water before it was used. This 
precaution was taken because the distilled water was very low in 
oxygen content and in it the pigment cells contracted. When 
oxygen was added no such contraction occurred. The details 
of the way in which the solutions were prepared are given under 
the respective heads. 

The experiments with the salts and the alkaloids were carried 
on in Syracuse watch glasses, which were kept covered to prevent 
excessive evaporation. They were uncovered only when actual 
observations were made. The amount of the solution used was 
about 10 cc. Experiments were performed in stender dishes 
of 50 cc. capacity as a check on the Syracuse watch glasses. 
There was no difference in the results. The experiments with 
volatile substances were carried on in wide-mouthed, glass 
stoppered bottles, with a capacity of 50 cc. All precautions 
were taken to prevent evaporation. 

Most of the experiments were carried on at room temperatures 
which varied between 69° and 72° F., although some were per- 
formed at the fish hatchery where the temperatures were from 
46° to 50° F. 

The experiments with the alkaloids and alcohols were started 
in solutions of 0.0001 per cent. The concentrations were in- 
creased in multiples of ten. 

The experiments were repeated ten to fifteen times for each 
solution tested. In many cases the experiments were repeated 
double the number, m order to eliminate all possible individual 
variation and errors. 



150 JOHN N. LOWE 

I wish, here, to express my chief indebtedness to Prof. 
M. F. Guyer, for his kindly criticism and suggestions during the 
progress of the work. To Prof. A. S. Loevenhart, I wish to 
acknowledge my appreciation of many courtesies extended. For 
the privilege and use of the fish hatchery and trout embryos, I 
desire to express my appreciation of the favor to Dean E. A. 
Birge and Superintendent James Nevin of the Wisconsin Fish 
Conmaission. 

Reactions to gases 

1 . Oxygen. The oxygen used in these experiments was chemi- 
cally pure. The pigment cells remained expanded in an atmos- 
phere of oxygen, and the fish lived indefinitely. 

The hydrogen used in these experiments was obtained by the 
action of chemically pm'e hydrochloric acid on Merck's highest 
purity zinc. The gas was passed through two towers of KOH 
and then through two towers of distilled water, of which one 
had red litmus, and the other blue litmus. The trout were in 
the fifth tower. 

The pigment cells contracted completely in four to six min- • 
utes when the embryos were exposed to hydrogen. If oxygen 
was substituted before the fish died the pigment cells expanded. 
If the oxygen was again replaced by hydrogen the pigment cells 
contracted. The results of these experiments show (1) that the 
absence of oxygen caused a contraction of the melanophores ; 
(2) that the oxygen is necessary for the mamtenance of the 
expanded pigment cells. 

3. Carbon dioxide. The carbon dioxide was generated through 
the intei-action of chemically pure hydrochloric acid on marble. 
The gas was purified by being passed through a tower of sodium 
bicarbonate and then through a tower of acidified lead acetate, 
and lastly through two towers of distilled water. 

The fish were exposed to water through which the carbon di- 
oxide was bubbling in a steady slow stream. The carbon dioxide 
produced a complete contraction of the pigment in two and one- 
half minutes. The time of contraction was the same for all the 



CHEMICAL AGENTS ON CHROMATOPHORE.S 151 

experiments performed. If an iiitense stream of oxygen was 
bubbled at the same time with the carbon dioxide, the pigment 
cells remained expanded. The proportion of the two gases which 
maintained the expansion of the melanophores was not de- 
termined. Briefly summarized the results prove that carbon 
dioxide produces a contraction of the pigment cells of trout 
embryos. The presence of oxygen antagonized the action of the 
carbon dioxide. 

Effects of distilled water 

The first experiments that were performed were to determine 
the effect of distilled water on the pigment cells of trout embryos. 
The normally expanded pigment cells contracted in ten to twelve 
minutes and the fish died usually in about twenty minutes — 
differing somewhat with the individual lots of fish. After an in- 
terval of ten to tMrty minutes, following the initial contrac- 
tion, the pigment cells began to expand. This secondary ex- 
pansion of the melanophores in no way equaled the normal ex- 
panded condition. The processes of the cells were short and 
blunt. This expanded condition lasted for a short period; then 
the walls of the melanophores began to break down and the cell 
contents, viz., the pigment granules migrated into the inter- 
spaces of the epidermal layer. Often the pigment cells disinte- 
grated without a previous expansion. Spaeth ('13) obtained 
essentially the same results with isolated scales of Fundulus in 
which the chromatophores (1) expanded, (2) contracted, (3) 
expanded a second time with a final degeneration. He did not 
try oxygenated distilled water. If 2 cc. of boiled tap water were 
added to 8 cc. of distilled water the results were the same. 
Then boiled tap water was tried and the pigment cells con- 
tracted in fourteen and twenty-two minutes. In distilled and 
boiled tap water through which oxygen had been bubbled the 
melanophores remained expanded and the fish lived indefinitely. 
The conclusion was obvious. It was oxygen want and not the 
absence of salts in the distilled water that caused the contrac- 
tion of ^he pigment cells and the death of the fish. 



152 JOHN N. LOWE 

Reactions to salts 

The problem of salt action is one of the most interesting 
within the scope of physiology and has wide applications. The 
relation of various salts to heart beat is a long debated question. 

Howell ('98), p. 49, is of the opinion "that the inorganic salts 
of the blood and liquids of the heart tissues especially of the 
calcium compounds, stand in a peculiar and fundamental rela- 
tion to' the initiation of the inner stimulus of the heart con- 
tractions." Loeb ('00 a, '00 b) believes that the sodium cations 
acting on the striped muscle to be the stimulating agents being 
counteracted by the ions of potassium and calcium. The posi- 
tion of Loeb is supported by Lingle COO). Benedict ('05 and 
'08) is of the opinion that the anion probably plays an important 
role in the action of salt solutions upon heart beat. 

Mathews ('04 a, '04 b, '05, '06) maintains that in the action 
of salt solutions on motor nerves, colloids, and sea urchin eggs, 
the ionic potential of the salt, which is the reciprocal of the 
solution tension, is an important factor in ionic action. R. S. 
Lillie ('11, '12 a, '12 b) working with the larvae of Ai-enicola 
and eggs of starfish, and McClendon ('10) on sea urchin eggs put 
forth the hypothesis that ionic action is due to the modification of 
the permeability of the plasma membrane. Loeb ('00 b) holds 
that ionic action is due to the formation of ion protein com- 
pounds, that is that the ions of the salt combine directly in some 
way with the protein molecules of the living protoplasm. True 
and Kahlenberg ('96) working with plants (Lupinus albus) be- 
lieve that the anion is unimportant in the toxic action of the 
salt. 

Spaeth ('13) working on the chromatophores in isolated scales 
of Fundulus heteroclitus concludes that the anion in potassium 
salts is of no importance in causing the initial contraction of the 
chromatophores, but that in the secondary expansion of the 
chromatophores the action of potassium is modified by the 
anions. On the other hand, the duration of the sodium expan- 
sion varies with the nature of the anion. 



CHEMICAL AGENTS ON CHROMATOPHORES 153 

The above opinions tend to show that the part played by ions 
in stimulation is by no means a settled question . In an attempt 
to gain further insight into the subject brook trout embryos were 
subjected to solutions of pure potassium and sodium salts. The 
results have been so promising that the work is being extended to 
numerous other salts. 

The salts used were of the purest of Merck's, Kahlbaum's and 
Baker's inanufacture. The solutions were made up in a 0.2 
molecular concentration with oxygenated distilled water. The 
solutions of the iodides which readily undergo decomposition 
were never older than thirty-six hours when used. 

The experiments were carried on in Syracuse watch glasses in 
about 10 cc. of the solution. At times small dishes of 25 to 50 
cc. capacity were used. 

1. Effects of potassium, salts. When the trout embryos are 
immersed m a 0.2 M. KI solution a rapid contraction of the nor- 
mally expanded chromatophores results within two or three 
minutes. They then appear as minute dots with no peripheral 
processes. In placing a similar lot into a 0.2 M K-2S04 equiva- 
lent solution the change does not occur as rapidly, being com- 
pleted in fifteen to twenty minutes. This at once suggested that 
there is a specific difference in the rate of contraction for potas- 
sium, varying with the anion. The experiments were extended 
to include the following neutral salts of potassium, viz., K0SO4, 
KCl, KBr, KNO3 and KI. Practically the first experiment 
showed that there was a distinct difference in the rate of con- 
traction varying with the anion. The rate and intensity of the 
contraction was most rapid in the order given (figs. 1, 2, 3, 4 
and 5). 

I> N03> Br> Cl> S04> 

In KI the contraction was complete before it had even begun in 
KCl or K2SO4. The experiments were repeated many times and 
as a check several of my colleagues were asked to come in and 
arrange the sets showing the greatest change. In all cases their 
arrangement was in the -above order. This clearly indicates that 
if contraction in the melanophore is specifically induced by the 



154 JOHN N. LOWE 

cation of potassium, it is unqualifyingly modified by its anion or 
the residual part of the undissociated molecules. 

Another interesting feature observed was that after a longer 
or a shorter interval after the first contraction there followed a 
peripheral expansion of the pigment cells (figs. 6, 7, 8, 9 and 10), 
that is, the pigment cells put out processes which became 
longer and longer as time went on but which never reached the 
original size they had before treatment with the potassium salt 
solutions. This expansion set in earlier in KI where the contrac- 
tion took place first, evidently the secondary expansion or pa- 
ralysis is reciprocal of the first contraction. The expansion is 
in the order of the first contraction (figs. 6, 7, 8, 9 and 10). 

I> N03> Br> Cl> SO4 

This peripheral migration of the pigment is in the nature of 
a paralysis. The paralytic state (depression) is soon followed by 
death of the pigment ceU. The walls of the pigment cell dis- 
integrate and the pigment granules flow into the interspaces of 
the body tissues. Death of the cells takes place often before 
the expansion is complete, and then premature disintegration of 
the pigment cells occurs. The condition or extent of the de- 
generation is dependent upon the 'physiological state' of the 
melanophores and the individual fish. 

The maintenance of the irritability of the melanophores fol- 
lowed the same order, correlated with this was the longevity of 
the fish. The fish lived the longest in K2SO4 and KCl. They 
died very rapidly in KI. 

The reactions varied with the concentration of the solutions, 
for in solutions of 0.1 M or less the changes were slightly slower. 
Molecular solutions gave no results but killed the fish imme- 
diately. 

3. Effects of sodnim salts. Here as in the potassium salts the 
embryos used had their melanophores expanded. It was ob- 
served that the neutral salts of sodium produced a contraction 
of the melanophores very slowly. In some instances the contrac- 
tion did not take place in 92 to 116 hours, especially in the solu- 
tions of Na2S04 and NaCl. The contraction in Nal was complete 



CHEMICAL AGENTS ON CHROMATOPHORES 155 

in five to forty-five minutes. It was confirmed by repeated ob- 
servation, that these contractions, slow as they may be for 
certain solutions (Na2S04 and NaCl), were in the following 
order : 

I> N03> Br> Cl> SO4 

A number of experiments were tried to determine if the sodium 
salts produced an expansion of the melanophores after the po- 
tassium salt contraction. The embryos were exposed to KCl 
from fifteen to twenty minutes when they were removed and 
rinsed in water to free them of the excess of KCl. They were 
now placed into the five neutral salts of sodium. The rate and 
degree of expansion was in the following order: 

S04> Cl> Br> N03> I 

The expansion was most rapid and complete in Na2S04 and NaCl. 
In Nal there was no expansion. 

The experiments were repeated with embryos that were not 
rinsed with water. The result was. the same as in those that 
were washed in water. If the melanophores are contracted with 
KI instead of KCl the results are the same. 

S04> Cl> Br> N03> I 

It is interesting to note here that no expansion of the mel- 
anophores occurred in the Nal solution. Is this because the 
sodium cations are inhibited in permeating the cell membrazie 
due to the presence of the dissociated iodine anions or some other 
factor? Are the cells permeable only to the iodine anions and 
not to the cations of sodium? Hamburger and von Lier ('02) 
claim that the blood corpuscles are permeable only for anions 
and are not permeable to the cations. If the expansion of the 
melanophore is specific for the sodium cation, it is overcome by 
the antagonistic action of the iodine anion, which produces a 
contraction. Nevertheless we must consider another factor, 
that is, the action exerted by the residual undissociated molecule 
which is present at all times in the solution. The expansion in- 



156 JOHN N. LOWTE 

duced by the sodium salts after a potassium salt contraction is 
followed by a contraction of the melanophores in the usual 
order. The position or order of the contraction was the same 
as for the expansion of the melanophores; but with one excep- 
tion where the NaNOs changed places with the NaBr. 

S04> Cl> N03> B,> I 

The extent to which the life of the fish and the irritability of the 
melanophores are preserved is possibly the function of the 
cation which is modified by the anion or the residual undisso- 
ciated molecule. 

3. Discussion. All these results seem to lend themselves to 
the interpretation that salt solution ha\'ing a common cation 
are modified by their anions or the residual undissociated mole- 
cule. This is clearly shown by the rate and degree of the con- 
traction of the melanophores by the potassium salts, where the 
contraction may be specific for the cation of potassium. Speath 
('13) p. 547 says in speaking of the action of potassium salts: 
"The time of tliis contraction (K) is the same for the five salts 
within the Umits of the variation of the individual scales. Since 
there is this common cation K+ in all five salts it seems prob- 
able that the initial effect (contraction) is specific for the K+ 
ions." My own results in the case of pigment cells of trout 
embryos are contrary to this conclusion. If contraction is spe- 
cific for the positive cation of potassium (K"^), it should be the 
same in rate and degree in all the salts of potassium. Since the 
rate and degree of the contraction are not the same for the five 
potassium salts (figs. 1, 2, 3, 4, and 5) it must depend on some 
other or some modifying factor which is responsible for this 
difference. 

A dissolved electrolyte conducts a current in proportion to the 
extent that it is dissociated or ionized. Its maximum conduc- 
tion will be at complete ionization which occurs at infuiite dilu- 
tion. Therefore the degree of the dissociation or the coefficient 
of dissociations can be obtained from the conductivity of solu- 
tion. The conductivity of an electrolyte divided by its num- 



CHEMICAL AGENTS ON CHROMATOPHORES 



157 



ber of gram equivalents in cms. is the molecular conductivity 
of the substance wTitten as A- However, the conductivity is at 
its maximum at infinitely dilute solutions, therefore the value 
A°= is taken as a measure of the total number of ions that are 
produced by the dissociation of one gram equivalent of the 
substance. Therefore d the degree of dissociation is directly 
proportional to the conducti\dty; thus we have the simple form- 
A 



ula 



The equivalent conductivity at infinite dilution 



for KCI is calculated to be 130.10. The equivalent conductivity 
of a two-tenth molecular KCI is 107.96 A 0.2 m. The degree of 

A 0.2 M 107.96 



dissociation at 18°C. is the ratio 



or 82.98 



A cc ' "' 130.10 
per cent. The values obtained in this way may be regarded 
only as approximate. The values are given in the following 
table. 



s^x 


i K2SO. 


KCI 


KBr 


KNO. 


KI 


Equivalent conductivity 
infinite dilution A "= 


at 


\ 1.32.8 


130.10 


132.30 


126, 50 


131 10 


Equivalent conductivity 
0.2 M dilution A 0.2 M 


at 


1 87.76 


107.96 


110.40 


98.74 


111.2 


Per cent or degree of dissocia- 


] 










tion a = A 0.2 M 
A == 




66.03 


82.98 


83.44 


78.05 


84.82 



A study of the table leads one to believe that the rate and the 
degree of the contraction are in some way correlated with the 
degree of dissociation of the salts. The lowest rate and degree of 
contraction was found in K2SO4, where the degree of dissociation 
is 66.03 per cent. The most rapid and complete contraction 
occurred in KI where the dissociation is 84.82 per cent. 

Potassium nitrate is out of place. It has a greater stimulating 
action than its degree of dissociation would indicate. It should 
fall between potassimn sulphate and potassium chloride. The 
possible explanation for this break in the series may be that the 

THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 1 



158 



JOHN N. LOWE 

TABLE 2 



S..T 


i NajSO, 


NaCl 


NaBr 


NaNOj 


Nal 


Equivalent conductivity at 
infinite dilution A °^ 


> 111.5 


108.99 


112.0 


105.99 


109.9 


Equivalent conductivity at 
0.2 M dilution A 0.2 M 


1 71.4 


87.73 


91.2 


82. 2» 


90.2 


Per cent or degree of dissocia- 


1 










tion a = A 0.2 M 


!• 64.03 


80.49 


81.43 


78.11 


82.08 


A °: 













nitrate anion exerts an independent action or it may form nitrites 
which are more active. 

In table 2 are shown the equivalent conductivities and degree 
of dissociation of the sodium salts. 

The values were calculated in the same manner as those for 
the potassium salts. Here, as in the potassium salts, the reac- 
tion of the melanophores was correlated with the degree of 
dissociation. 

There are two reactions of the melanophores which are char- 
acteristic of the potassium salts: (1) a primary contraction, 
(2) an expansion which is the sign of death or degeneration of 
the cell. The cell wall breaks down and the pigment granules 
escape into the surrounding tissues. The degree of the cytolysis 
is directly proportional to the degree of dissociation of the salt. 
In sodium salts we have two specific reactions : ( 1 ) the expansion 
and maintenance of the expansion for a certain period of time, 
(2) a slow contraction. The two reactions of sodium salts occur 
in an inverse order to those of the potassium salts, where con- 
traction is followed by a cytolytic expansion. The contraction 
in sodium salts is not followed by a cytolytic expansion, but the 
disintegration takes place directly from the contracted pigment 
cell. This contraction in sodium salts is directly comparable 
to the cytolytic expansion observed in potassium salts, for both 
of these stages indicates the death of the pigment cell. 

A. P. Mathews ('OG) suggested that it is the ionic potential 
of the ions, and not the difference of voltage between the plate of 



CHEMICAL AGENTS ON CHROMATOPHORES 



159 



a metal and any solution of its salts, but rather the difference 
in pressure between a single ion and a single atom of the metal 
that determines the chemical action of the ions. Since solution 
tension is a measure of the difference in potential between the 
solution which contains a known amount of the ions of the metal 
and the metal itself, it is also the difference between the tendency 
of an atom of the plate to become an ion. When applied to 
living protoplasm the metal plate is replaced by the protoplasm. 
The value varies with the amount of electrolytic dissociation 
and the kind of plate present. 

The solution tensions in volts of elements in normal ionic 
solutions. 

CI 1.694 

K 2.92 Br 1.270 

Na 2.54 1 0.797 

NO3 2.229 

The ionic potential is the reciprocal of the solution tension. 
Ionic potential is the tendency of any ion in any concentration 
of solution to change into an atom of its metal. 

The ionic potentials of the ions of metals in volts are: 

Cl 1.694 (?) 

K 2.92 (?) Br 1.270 (?) 

■Na 2. ,54 (?) 1 0.797 (?) 

NO3 2.229 (?) 

Mathews ('06) shows that the dissolving power of the salts 
of sodium and potassiun for edestine, a globulin of the hemp 
seed is in some way correlated with the ionic potential. 



S...X 


lO.VIC POTENTI.\L 


CENTIMETERS RE- 

QCIREDTO DISSOLVE 

ONE GRAM or 


Kl 


-2.123 

-1.65 

-1.226 

-1.743 

-1.270 

-0.846 




5.7 


KBr 


10 


KCl 


15 1 


Nal 


5 7 


NaBr 


9 3 


NaCl 


12 8 







160 JOHN N. LOWE 

The more negative the value for the ionic potential the greater 
the solvent power of the salt for edestin. The negative value in 
potassium is much greater than that in the sodium. In the table 
we observe that it takes like amounts of the iodides and less of 
the other sodium solutions to dissolve the edestin. However, we 
should expect it to take less of the potassium salts than it does 
of the sodium. I find this to be true for the pigment cells of 
trout, where the potassium salts cause the contraction of the 
pigment cells more rapidly than do the salts of sodium. Un- 
fortunately the solutions tensions for sodium and potassium are 
more or less indefinite which makes the results obtained for the 
salts of these metals incomparable. The ionic potential is not 
determined directly, but calculated only, thus making the ex- 
planation more difficult. 

The results obtained in experiments on the action of salts on 
the pigment cells of trout are explicable on three assumptions; 

(1) that it is the antagonistic action between anion and cation, 

(2) that it is the Independent action of the cation, (3) that the 
reaction is modified by the residual undissociated molecule. 

The antagonistic action between anions and cations has been 
postulated by Mathews ('06), Benedict ('05, '08), and W. Koch 
('09). The increased action of different salts having the same 
cation have been observed in different tissues. Loeb ('99) pro- 
duced a better rhythmical contraction in striped muscle wdth 
NaT than he did with NaCl. Zoethout ('04) confirmed this ob- 
servation, and extended it to KI which increased the muscle 
tone more than KCl. Benedict ('08) concluded that "the direct 
production of rhythmic activity by means of a salt's action upon 
heart muscle is due to the anion of the salt, while the chief 
function of the cation is apparently to maintain such a tone of the 
heart muscle that it will respond to the stimulus furnished by 
the anion." Mathews ('02) has shown that the presence of 
iodine, bromine anions stimulated the motor nerve more power- 
fully than the chlorine anion. Speath ('13) observed that the 
cytolytic expansion of the melanophores in potassium solutions, 
varied with the anions, but he did not note a difference in the 
rate of the primarj'^ contraction of the melanophores in the 



CHEMICAL AGENTS ON CHROMATOPHORES 161 

different potassium salts. In sodium salts the expansion of 
contracted melanophores varied with the anion, and the con- 
traction following this expansion was correlated with the anions. 
In neutral salts of potassium there are two constant results 
produced on the pigment cells of trout; (1) a contraction of the 
pigment cells, (2) a cytolytic expansion. The times for each 
varied with the anion. If the antagonism existed between the 
potassium cations K +, and the negative anions SO4-, Br-, 
CI - , N03 - , I - , it was the least effective in KI and most potent 
in K2SO4. The order of contraction and expansion was 

I> N03> Br> Cl> SO4 
In sodium salts there were two characteristic reactions, (1) an 
expansion, (2) a contraction. The rate and degree of the ex- 
pansion of the melanophores was greatest in NasSOi and least 
in Nal. The rate of contraction was rapid in Nal and least in 
NasSOi. The order of the expansion was 

S04> Cl> Br> N03> I 

The contraction rate of the pigment cells was inverse to the 
above. 

I> Br> N03> Cl> SO 

The cationic action was modified by the nature of the anion. 
This anionic order was observed by Paul and Kronig ('96) on 
the disinfecting power of mercuric salts of chloride bromids and 
cyanide. Mathews ('06) has shown for the eggs of Fundulus 
heteroclitus that the fatal dose varied with the anion. Loeb and 
Cattell ('15) have shown that the hearts of Fundulus embryos, 
previously poisoned by KCl, and recovered by sodium salts 
was an anion effect inasmuch as it increased with the anion, 
apparently in agreement with Hardy's rule (ion effect = ex- 
ponential function of the valency) for the acetate was much 
more efficient than the chloride. 

2. That it is the cation of potassium or of sodium that causes ■ 
the reaction of the pigment cells of trout embryos. 

Loeb ('10, '12) and Loeb and Wasteneys ('11a and 'lib) 
maintain that there is an antagonism between the sodium cation 



162 JOHN N. LOWE 

Na + and the potassium cation K + and not between the po- 
tassium cation and the chlorine anion K + CI — . This is sup- 
ported in part by the foregoing experiments on the jiigment 
cells of trout embryos. The pigment cells are expanded in 
sodium salts after a potassium salt contraction. But this is not 
true of all the salts of sodium. If the pigment cells are con- 
tracted in KCl or KI and are now placed in Nal there is no ex- 
pansion. Apparently there is an antagonism between the 
dissociated anions of (CI — and I — ) and the sodium cation 
(Na +) for from the conditions of the experiment we should get 
an expansion. It is probable that Loeb underestimated the 
antagonism between the positive ions of K -|- and Na 4- and 
their negative ions CI — . The longevity of the fish is better 
protected in sodium salts than in potassium salts. But again 
some of the sodium salts are more protective (Na2S04 or NaCl) 
than others (Nal). That the potassiuin and sodium cations do 
exert some such modifying action is undeniable, but to say that 
it is independent of its anion is not warranted by the facts at 
our command. 

3) That it is the residual undissociated molecules in the 
solution that modify the action of the salt. 

In 0.2 M, KI the degree of dissociation is much greater than 
in an equivalent 0.2 M, solution of K2SO4. Correspondingly KI 
initiates more intense responsiveness of the pigment cells than 
does K2SO4. The rate and degree of the reactions of the pigment 
cells decline as the number of the undissociated molecules in- 
creases. In the potassium salts the primary contraction and the 
expansion vary with undissociated molecule, thus, 

S04> Cl> Br> NO, I 

The degree of dissociation for 0.2 M solutions are 

66.03 82.98 83.44 78.05 84.82 

The fact that the nitrate is out of place was mentioned before. 
As already stated, this may be due to the independent activity 
of the nitrate, which may break down to form a nitrite. 

In sodium salts the dissociation percentages are slightly less 
than in potassium salts. The rate of contraction is much slower. 



CHEMICAL AGENTS ON CHROMATOPHORES 163 

The fish Uve longer in sodium solutions, and the pigment cells 
retained their irritability longer. The pigment cells expand if 
transferred after they are contracted in potassium salts. Is 
this reaction specific for the sodium ion or is it due to the in- 
creased number of undissociated molecules in sodium solution? 
It cannot be positively concluded whether this difference in 
residual molecules is enough to account for the difference in the 
rate of contraction of the melanophores in sodium and potas- 
sium salt solutions. The ascribing of the principles of salt ac- 
tion to the anion or cation without the consideration of the 
residual undissociated molecule is just as out of proportion in 
the field of physiology as to say that the undissociated alkaloids 
and other substances have no action. 

Reactions to alcohols 

Whether or not alcohols have a stimulating action is a much 
debated question. The Schmiedeberg school of pharmacolo- 
gists maintains that alcohols produce no primary stimulation of 
the central nervous system. According to this view the giving 
alcohol to a mammal, if followed by an increased muscular ac- 
tivity, is said to be due to the depression of the cerebral centers, 
thus removing the restraint from the motor areas. Binz and 
his followers hold to the view that alcohol first stimulates and 
then depresses the nerve cells. 

The literature on the pharmacological action of alcohol on the 
heart and other tissues is very extensive, but to my knowledge 
there are no records of any attempts to determine its action on 
the melanophores. Whatever action the alcohols exert on the 
melanophores will not settle the question whether alcohols 
stimulate or depress the nervous system, as the melanophores in 
the very nature of their origin and structure must be looked upon 
as specialized mesenchymal cells. While it is not at all improb- 
able that the general facts observed with melanophores may^ be 

' After these experiments were completed Spaeth 1916 published results, 
where he subjected isolated scales of Fundulus to vapors of alcohol, ether and 
chloroform and always obtained a contraction of the melanophores, and larger 
amounts of these vapors inhibited the contraction. 



164 JOHN N. LOWE 

true also of other tissues, I refrain from applying the results to 
such an interpretation. The literature is used in a compara- 
tive way, but not in the sense that the results obtained with 
melanophores are directly comparable. 

Ten per cent stock solutions of methyl (Sp. G. O. 796), ethyl 
(Sp. G. O. 796-800) propyl (Sp. G. O. 8066) alcohols of Merck's 
manufacture were made up with oxygenated distilled water. The 
dilutions were made from these stock solutions with oxygenated 
distilled water. The experiments were carried on in glass stop- 
pered bottles of 75 cc. capacity. All work was done at room 
temperature of 18° to 20°C. 

1. Methyl alcohol. Overton ('01) showed that methyl alco- 
hol has a less powerful narcotic action on tadpoles than ethyl 
alcohol. Vernon ('11) confirmed that the same was true in the 
•depressing action of methyl alcohol on the heart muscle of a 
turtle's heart. 

Young brook trout embryos of the same age and condition were 
subjected to the action of the respective alcohols of the various 
concentrations. The contraction of the pigment cells was taken 
as the criterion of stimulation, the relaxation (expansion) as 
that of a depression. 

Ethyl alcohol in solutions of 1.6 to 2.5 per cent produced a 
complete contraction of the pigment cells. Methyl alcohol of an 
equal concentration did not cause a contraction. In a 3.5 per 
cent solutiort there was a slight retraction of the pigment cells, 
but the contraction was not complete. A 4.5 per cent solution 
produced a complete contraction of the melanophores. Solu- 
tions of 5 per cent to 5.5 per cent produced a slight contraction 
of pigment cells This partial contraction was followed by an 
immechate expansion. If embryos in which the melanophores 
were just contracted in a 0.005 per cent strychnine solution were 
subjected to 5 per cent to 5.5 per cent methyl alcohol the pig- 
ment cells expanded. In 7 per cent to 10 per cent solutions of 
methyl alcohol there was no visible change in the expanded 
melanophores. 

Thus it may be concluded that (1) methyl alcohol in high con- 
centration acts as a depressing agent, (2) in medium concentra- 



CHEMICAL AGENTS ON CHROMATOPHORES 165 

tion it has a stimulating action, and (3) in very weak solution it 
has no effect on the melanophores of trout embryos. Methyl 
alcohol has a less pronounced stimulation action than ethyl 
alcohol on pigment cells of trout It was necessary to double 
to concentration so as to bring about reactions in any way com- 
parable to those produced by ethyl alcohol. The action of 
methyl alcohol was less striking and the stages of stimulation 
and relaxation were slower in appearing than in ethyl alcohol. 

2. Ethyl alcohol. — When trout embryos were exposed to weak 
solutions (0.01 per cent to 0.8 per cent) of ethyl alcohol, no change 
took place in the pigment cells. The embryos did not show any 
signs of depression and appeared perfectly normal. In solutions 
of 1 per cent to 1.5 per cent the embryos became more restless 
and the pigment cells exhibited a partial contraction. In con- 
centrations of 1.6 per cent to 2.5 per cent of ethyl alcohol the 
fish became more active, the pigment cells showed a complete 
contraction; while in solutions of 3.0 per cent to 4.5 per cent 
they showed a transitory contraction, followed by an expansion. 
This result could be very easily overlooked. In 6 per cent to 
10 per cent solutions the trout embryos died rapidly in from fif- 
teen to twenty-five minutes, and there was no contraction of the 
pigment cells. If embryos that had their pigment cells con- 
tracted in the 2 per cent solution were transferred to a 7 per cent 
the pigment cells expanded rapidly. 

If the embryos in which the pigment cells were contracted were 
transferred to a 4.5 per cent to 6 per cent solution an imme- 
diate expansion resulted This expansion was due to the de- 
pression caused by the high concentration of the alcohol, which 
was far beyond the maximum threshold of stimulation. When 
the fish which had their melanophores contracted in a 2 per cent 
solution were placed in a 0.5 per cent solution they expanded. 
Here the dilution ofHhe alcohol was below the threshold stimu- 
lus. If embryos that were exposed to 7| per cent solution for 
an interval of four to six minutes, were placed in water or very 
weak alcohol, there was observed a contraction of the pigment 
cells which was of a very short duration. This result was no 
doubt due to the washing out or the dilution of the alcohol within 



166 JOHN N. LOWE 

the tissues, to the threshold stimulus and as the process of 
dilution continued the point was reached where the concentration 
fell below the threshold and a relaxation (expansion) of the 
melanophores occurred. After a complete recovery of the em- 
bryos from the effects of the alcohol the pigment cells reacted 
normally to other stimuli. 

These results show clearly that very weak solutions of ethyl 
alcohol do not have any effect on the pigment cells of the trout 
embryos. This is in harmony with the work of Kobert ('82) 
on the frog's muscle, Lee and Salant ('02) on the gastrocnemius 
muscle of the frog, and Carlson ('06) for the heart muscle and 
heart ganglion of Limulus, all of whom observed that weak or 
very weak solutions of ethyl alcohol had no stimulatory action. 
Ethyl alcohol in contractions of 1.3 per cent to 2.5 per cent water 
shows a decided stimulatory action on the pigment cells of brook 
trout embryos. This is in accord with results of others on the 
primary stimulation of ethyl alcohol. Pickering ('95) has shown 
that alcohol excites the embryonic heart muscle of the chick. 
Scheffer ('00) has observed that in the frog's gastrocnemius when 
it was treated with alcohol the capacity for work was increased. 
If the muscle was curanized the stimulating effect of alcohol 
was nil. 0. Loeb ('05) has noted that in solutions of 0.13 to 
0.3 per cent that the action of the isolated mammaUan (cat) 
heart was augmented. Wood and Hoyt ('05) have shown that 
small amounts of ethyl alcohol increased the force of the heart 
beat in the frog, snake, tortoise, and turtle. Lee and Salant 
('02) have demonstrated that in medium concentrations of ethyl 
alcohol there was an increased rate of contraction and relaxa- 
tion in frog's muscle (gastrocnemius). Carlson ('06) has ob- 
served that for the heart nmscle and heart ganglion of Limulus, 
alcohol stimulated. Vernon ('10) has shown that alcohol has an 
excitatory effect on the isolated heart of the turtle (Emys). The 
('02) observed a marked increase in the number of contractions 
of the bell of the Medusa Gonionema in ethyl alcohol of 0.5 
to 0.25 per cent. 

In a strong concentration of 4.5 per cent there was a marked 
depression or an expansion of the pigment cells. In this con- 



CHEMICAL agents" ON CHROMATOPHORES 167 

centration there was no primary stimulating period observed. 
If it is to be found, it may be so short as to be very easily over- 
looked. Alcohol in large amounts decreased the rate of contrac- 
tion in the gastrocnemius frog's muscle, Lee and Salant ('02). 
Romanes ('77) found that strong solutions of ethyl alcohol pro- 
duced increased and spasmodic contraction of the medusa bells 
of Sarsia (sp.) and Tiaropsis (sp.). These were followed by a 
depression. 

Lee ('02) observed that in solutions of ethyl alcohol of a greater 
concentration than 2 per cent the contractions of the bell of the 
medusa, Gonionema, were much reduced in volume and in 
number. Dogiel ('77) has shown a depression in the heart 
rhythm of Corethra plumicornis. Vernon ('10) observed that 
large doses of ethyl alcohol depressed the rate and volume of 
the contraction of a turtle's heart (Emys). 

3. Propyl alcohol. Weak solutions of propyl alcohol 0.01 per 
cent to 0.04 per cent did not effect the melanophores. In a 0.06 
per cent there was a noticeable contraction of the pigment cells. 
Solutions of 0.08 per cent to 0.125 per cent produced a rapid and 
complete contraction In 0.7 per cent to 1.3 per cent the contrac- 
tion was only temporary, and was followed by an immediate 
relaxation of the pigment cells. A 1.5 per cent to 2 per cent 
produced no visible change in the expanded melanophores, and 
when embryos with contracted melanophores were exposed to 
the solution the melanophores expanded. In these concentra- 
tions there was observed a marked disintegration (cytolysis) 
of the cells. Higher concentrations (2.5 per cent to 4 per cent) 
killed the embryos without inducing any change in the expanded 
pigment cells. Contracted cells exposed to these solutions ex- 
panded instantaneouslj^ and after this response gave no reactions 
to other stimuli. 

It is obvious from these results that the stimulation of the 
pigment cells by propyl alcohol begins in solutions of lower con- 
centrations than it does in ethyl and methyl alcohol. It will be 
seen that my results for methyl, ethyl, and propyl alcohols are 
in perfect agreement with the results on the toxicity of the 
above alcohols of other investigators. 



168 



JOHN N. LOWE 



Joffroy and Serveaux ('95) studied the toxicity of alcohols 
on maniinals by intravenous injections. Bear ('98) introduced 
the alcohol directly into the stomach of the mammals. Picaud 
('97) placed fish and amphibians in the solutions of the alcohols 
and in this way determined the toxicity of the alcohols. Brad- 
bury ('99) andCololian ('01) used fish, Overton ('01) on tadpoles 
employed the same method in their investigations. Wirgin ('04) 
determined the concentrations al which the various alcohols in- 
hibited the growth of Micrococcus pyogenes aureus. He also 
investigated the laking power of the alcohols on the red corpuscles 
of the rabbit. Vernon ('11) studied the depression of an iso- 
lated tortoise heart by the alcohols. In table 3 the toxicity 
of ethyl alcohol is taken as unity and the values given are the 
comparative toxicities of the other alcohols. The values are 
only approximate. 













TABLE 3 






























, f. 




. « F- 




























■'i 


< 


ta 


1 


i 




J 


«i 


^a 


< a " H >. 


i - H B 




1^ 




< 


< ^ 


s" 


o 


3^ 

£ < 


S8 


> 


imi 


llhl 


Methyl 


n 4fi 


n 8 


n 67 


1 n 


1 1 


73 


n 73 


n S4 


n 7'' 


0.45 


0.55 


Ethyl 


1 n 


1 


1 n 


1 n 


1 n 


1 


1 n 


1 


1 


1 


1 




3.5 


2.0 


2.0 


1.0 


3.6 


2.0 


1.5 


2.1 


2.1 


2.0 


3 







The stimulation level is lowest in methyl alcohol (4.5 per 
cent); next is ethyl alcohol (1.6 per cent to 2.5 per cent); and 
lastly propyl alcohol (0.08 per cent to 0.125 per cent). This is 
in harmony with the results of other investigators on the toxi- 
city of alcohols where it was found that methyl was less potent 
in bringing about narcosis, and the potency increased for the 
other alcohols directly with the molecular weight. It was shown 
by Baer ('98) that the toxicity of the alcohols varied directly as 
their boiling points. Meyer ('99) and Overton ('01) discovered 
that the narcotic action of the alcohols varied with their solvent 
power for fats or lipoids. It may be suggested that in addition 
to the above physical factors involved in the action of the alco- 
hols, that the dielectric constant of the alcohols probably plays 



CHEMICAL AGENTS ON CHROMATOPHORES 



169 



an important part in their action. It was observed that the 
greater the dielectric constant of the alcohols used the lower the 
stimulating or depressing power, and conversely the lower the 
dielectric constant the more striking were the reactions. What- 
ever may be the relation of these physical factors of the alco- 
hols in stimulation or depression, their chemical structure must 
not be overlooked ; for as the length and complexity of the chain 
in monohydric alcohols increases so does the strength of their 
action. 



...COHO.S 


MOLECULAR 


CONST.\NT 
AT 20°C 


BOILING POINT, °C. 


POWER — ETHYL 

ALCOHOL TAKEN 

AS 1 


Methyl 

Ethyl 


32.03 
46 05 
60.06 


31.2 

25.8 
22.0 


65.7 
78.4 
97.4 


0.45 
1.0 




2.0 







Reactions to alkaloids 

The study of the action of di'Ugs on the pigment cells of trout 
was undertaken with a threefold purpose, viz., to compare the 
action of drugs on the pigment cells with that of other tissues; 
second, to determine if possible the controlhng mechanism of the 
pigment cells, and third, to see if the drugs had a specific action 
on the pigment cells. 

The literature on the pharmacology of the pigment cells of fish 
is not very extensive. The earliest historical record of experi- 
ments on the action of drugs on the pigment cells is that of Redi 
(1664), who observed that eels which died in a tobacco decoction 
were light in color. Pouchet (76) observed that Gobius niger 
changed in color when placed in strychnine. Morphine, qui- 
nine, and santonin had no effect. Lode ('90) concluded that 
curare destroyed the nerve endings of the pigments cells of 
trout (Salmo fario). von Frisch ('11) found that chloral hy- 
drate contracted the pigment cells of the minnow and crucion. 
He also concluded that the action of cocaine was through the 
central nervous system. 

The pigment cell may be stimulated or depressed by the drug 
acting: 1) on the pigment cell in such a way as to increase or 



170 JOHN N. LOWE 

decrease its irritability; 2) on the nerve endings leading from the 
gangha controlhng the ]3igment cells; 3) on the central nervous 
system. I have no direct evidence to offer which will enable us 
to determine which of these or combination of these three fac- 
tors are operative in the action of the drugs on the pigment 
cell, for I was miable to separate the nervous and pigment cell 
tissues for experimental purposes. It is obvious that large 
doses have no selective action. At certain optimal concentra- 
tions all the drugs show a selective action on the pigment cells 
or their controlling mechanism. This selective action of drugs 
on the mechanisms of the pigment cells will further our knowl- 
edge as to their function. 




Fig. 1 A normal brook trout embryo showing the general alignment of the 
body. 

In interpreting my results I have given special emphasis to 
their relation in a comparative way to the observations of other 
observers on various vertebrate and invertebrate tissues. This 
comparative method makes the results easier of interpretation 
and is less liable to lead to an erroneous conclusion. 

The drugs used were all of INIerck's manufacture. They were 
dissolved in oxygenated distilled water. The stock solutions 
were made up from 0.25 per cent to 0.5 per cent. Dilutions were 
made from these solutions. The experiments were carried on in 
Syracuse watch glasses in 10 cc. of the solution. These re- 
sults were checked by experiments in small stender dishes of 
50 cc. capacity. The conclusions are based on experiments re- 
peated ten times in 1913 and again in 1914 another series of ten 
was tried. Five to ten anmials were used at one time in each 
dilution. The trout embryos used were from four days to two 
weeks after hatching. In no case were the mdividuals of the 
different ages mixed. 

' Figures 1, 2, and 3 were drawn by Miss H. J. Wakeman. 



CHEMICAL AGENTS ON CHROMATOPHORES 171 

Other experiments are in progress to determine the action of 
drugs on pigment cells isolated from the nervous system. 

1. Strychnine. In 0.5 per cent oxygenated solution death re- 
sulted without primary stimulation of the pigment cells. In 
0.05 per cent strychnine solution the results were the same. 
Solutions of 0.005 per cent strychnine caused a contraction of the 
pigment cells rapidly, the contraction was complete in five min- 
utes. There was a remarkable thing observed in this concen- 
tration of strychnine. The irritability of the fish was increased 
to a high degree. The fish went into typical strychnine spasms. 
The head was thrown backward and the tail curved upward and 
forward, describing a half circle (as shown in text fig. 2). A 




Fig. 2 Showing a brook trout emljryo in a typical opisthotonos response in 
0.005 per cent strychnine. 

passing shadow over the disk brought on a new spasm. Shadows 
in rapid succession increased the concavity backward. If the dish 
was tapped very lightly the same responses occurred. This 
period of heightened excitability lasted from ■ eight to twelve 
minutes. Durmg this interval the pigment cells remained con- 
tracted (fig. 13). As this convulsive period disappeared, the pig- 
ment cells expanded (fig. 14). This expansion showed that the 
depression and paralysis of the pigment cell controlling mecha- 
nism had occurred. In 0.0005 per cent the pigment cells were 
contracted in ten minutes. No convulsions were observed in this 
concentration. In weak solutions of 0.00005 per cent to 0.000025 
per cent no contractions of the melanophores was produced. 



172 JOHN N. LOWE 

Pouchet ('76) observed that the pigment cells of Gobius niger 
contracted m strychnine solutions. Romanes ('77) noted that in 
the medusa Sarsia (sp.) the swimming motions were much 
accelerated by strychnine, also that convulsions occurred in this 
and three other forms — Cyanaea capillata, Tiaropsis indicans, 
and Tiaropsis diademta. Hedborn ('99) has shown that 
strong doses of strychnine augment the beat of the isolated mam- 
malian heart (cat). Dogiel ('77) demonstrated a slight increase 
in the rate of the heart beat of Corethra larvae. Pickering ('93) 
observed that weak solutions of strychnine had a primary stimu- 
lating action on the heart muscle of an embryonic chick. Carlson 
('06) has found that strychnine in very weak concentrations had 
a distinct stimulatory action on the heart ganglion of the Limu- 
lus heart. Stronger solutions produced augmentation followed 
by paralysis. He was unable to note any primary stimulation 
on heart muscle. Laurens ('15) observed that if a drop or two 
of a 1 per cent solution of strychnine was injected into the 
body cavity of Amblystoma larvae the pigment cells contracted. 

All the above experiments on other tissues show that strych- 
nine has a primary stimulating action and especially on the 
motor ganglia. From the evidence of Ballowitz ('93) who dem- 
onstrated that the pigment cells of fish have a connection with 
the nervous system, and from the fact that strychnine stimu- 
lates the nervous system, we are warranted in concluding that 
strychnine acts directly on the nervous mechanism controlling 
the melanophores of trout embryos, rather than on the melano- 
phores themselves. The seat of strychnine poisoning is in the 
spinal cord, therefore, the melanophores of trout embryos are 
in all probability controlled in part by the spinal nervous 
system. 

2. Picroioxin. Picrotoxin is used as a fish poison. It pro- 
duces a medullary stimulation and ultimately results in death. 
When trout embryos are exposed to a 0.25 per cent solution of 
picrotoxin the pigment cells contract rapidly. The contraction 
is complete in two to five minutes. The contraction remains 
for forty-eight to sixty-four hours, if the fish are kept in this 
solution. The fish live in 0.25 per cent solution for one hundred 



CHEMICAL AGENTS ON CHROMATOPHORES 173 

and twenty-six hours. There is no convulsive period. In a 
weak sokition of 0.025 per cent of pictoroxin the contraction is 
shghtly less rapid, and lasts indefinitely (fig. 11). 

When the tail is cut away the pigment cells in the tail portion 
expand (fig. 12). They remain expanded for six hours and then 
degeneration sets in. The melanophores in the anterior or head 
end remain contracted. The contraction continues for eight to 
twelve hours and then disintegration of the pigment cells occurs. 
This justifies the conclusion that the reactions of the pigment 
cells of trout embryos are in some way controlled by the higher 
nerve centers. 

If the pigment cells that are contracted in picrotoxin are ex- 
panded in 0.2 M. NaCl and are now placed in picrotoxin the 
contraction is much slower than the first time. The sodium 
chloride seems to counteract the action of the picrotoxin. 

3. Morphine. In embryos exposed to 0.5 per cent solution of 
morphine hydrochloride the pigment cells remain expanded. 
In a 0.12 per cent most of the pigment cells were expanded but 
there were a few isolated areas that, showed a contraction. After 
an exposure of three hours these isolated areas ' of contracted 
pigment cells had increased. In a 0.06 per cent solution of 
morphine the result was the same. In a 0.012 per cent solution 
no change occurred, all the pigment cells remained expanded. 
There was no contraction of the pigment cells in a 0.005 per 
cent solution. Pigment cells contracted by picrotoxin, potas- 
sium iodide or strychnine were expanded by morphine. Ac- 
cording to Pouchet ('76), morphine did not cause any change 
in the pigment cells of Gobius niger. Romanes ('77) has found 
that in Aurelia aurita morphine had a highly depressing action. 
Pickering ('93) found that morphine £«cetate depressed the 
action of the heart muscle of embryo chicks. Cushny ('10) says 
that the action of morphine on the central nervous system is a 
mixture of stimulation and depression which are not equally 
marked throughout the system; also, "there is a selective action 
on the medulla oblongata in which certain centers are entirely 
paralyzed before neighboring ones undergo any distinct modifi- 
cation." Waller ('96) found that morpliine applied directly to 
the nerve had but little effect on its irritability. 

THE JOURNAL OP EXPERIMENTAL ZOOLOGT. VOL. 23, No ] 



174 JOHN N. LOWE 

The explanation for the localized areas of contracted pigment 
cells may depend upon the selective action of morphine upon the 
nervous system. 

The foregoing experiments support the conclusion that the 
pigment cells are controlled by the medulla or the spinal cord. 
It is probable that the locaUzed areas of expanded and contracted 
pigment cells are in direct response to the mixture of stimulations 
and depressions caused by the action of morphine on the me- 
dulla. Or if the pigment cells are controlled by the reflex irri- 
tability of the spinal cord which may be depressed for a period 
and then may be followed by an increased irritability. On the 
latter hypothesis all the pigment cells should contract during 
the heightened irritability or expand dm-ing the diminished 
irritability; but since this is not the case it is probable that all 
the regions of 'the spinal cord are not involved at the same time. 

U- Caffeine. In embryos exposed to a 0.2 per cent to 0.25 per 
cent solution of caffeine citrate no change occurred in the pig- 
ment cells. The animals died in a much distorted condition. 
The pigment cells disintegrated in two hours. A 0.05 per cent 
solution of caffeine citrate caused the pigment cells to con- 
tract in 4.25 minutes. There was a pecuhar twitching of the 
muscles which lasted twelve minutes. A depression occurred in 
foirrteen minutes. The pigment cells expanded very rapidly. 
In 0.025 per cent caffeine citrate solution contraction of the pig- 
ment cells took place in 5.25 minutes. The depression or paraly- 
sis was elicited in thirty minutes in some, whUe in others it took 
forty-five to sixty minutes. A solution of 0.005 per cent caffeine 
citrate caused no contraction of the pigment cells in two and 
one-half hours. 

The convulsions observed were quite similar to those that 
occurred in strychnine. In caffeine the responses to shadows 
were absent. If the dish was jarred the reactions were weaker 
and lasted a short interval. These reactions occurred in solu- 
tions ten times as strong as in strychnine. The response was not 
opisthotonus, but the head was drawn toward one side and the 
tail toward the other. There was no difference in the sides to 
which the curvature occurred (as shown in text fig. 3). The 



CHEMICAL AGENTS ON CHROMATOPHORES 175 

animal was in the form of the letter S. The convulsive period 
lasted a short time and gave from one to six spasmodic reactions. 
The pigment cells remained contracted during this period. As ' 
the convulsive tremors gave way to a complete paralysis the 
pigment cells expanded. The convulsive period and the con- 
traction of the pigment were simultaneous. Weak solutions of 
0.0005 per cent had no effect on the trout embryos or their pig- 
ment cells. 

If the embryos are removed from a 0.05 per cent caffeine citrate 
solution during the period of convulsions, and if the poison is 
washed out rapidly there is a complete recovery. The 'pigment 
cells expand normally. If removed during paralysis after con- 




Fig. 3 Showing ii brook trout embryo in a typical caffeine convulsion. 

vulsions the fish may recover very slowly or not at all. In 
weaker solutions of 0.01 per cent to 0.025 per cent there are no 
convulsions, but only a contraction of the pigment cells; there 
is a complete recovery when they are placed in water. 

Carlson ('06) has shown that caffeine caused a primary aug- 
mentation in the heart muscle and primary stimulation of the 
heart ganglion of Limulus. Hedborn ('99) observed that caf- 
feine stimulated the isolated mammalian heart (cat). Pickering 
('93) observed an increase in the number of heart beats in the 
embryo chick's heart, and concluded from his work that it is 
not necessary to introduce a nervous hypothesis to explain the 
action of caffeine. Romanes ('77) has found in Sarsia (sp.) 
exposed to a sea water saturated with caffeine there was a great 
increase of the contraction and at the same time a diminution 



176 JOHN N. LOWE 

of the potency of the beat. Soon the pulsations became of a 
fluttering nature and spontaneous movements ceased. 

In the pigment cells of trout there is a stimulation which is 
at its height during the convulsive period. This is soon followed 
by a paralysis and an expansion of the pigment cells results. 
There is a direct resemblance in the results obtained with caffeme 
and strychnine, in that the reflex irritability is remarkably in- 
creased. The pigment cells contract in both instances during the 
convulsive period. There is a similarity in the results on the 
pigment cells of trout and the work of other investigators on 
other tissues. CafTeine may act directly on the pigment cells as 
it does on muscle (Pickering, '93, Carlson, '06), or it may 
stimulate the reflex centers in the medulla and spinal cord, 
which give off the fibers which control the pigment cells. 

5. Curara. In very strong solutions of curara of 2 per cent 
to 1 per cent, a few pigment cells contracted. When trout em- 
bryos were exposed to a 0.5 per cent solution they moved about 
rapidly for eight to ten minutes. In one case one showed a 
complete contraction of the melanophores in thi-ee minutes 
while the other nine fish in the same lot showed no change. 
The one that showed this contraction had its pigment cells com- 
pletely expanded in thirteen minutes. In 0.25 per cent solution 
there occurred a partial contraction of the pigment cells. The 
pigment cells along the lateral line were not contracted. The 
fish died in an hour and were covered with a colorless jelly- 
like slime. In the following dilutions of curara, viz., 0.05 per 
cent, 0.025 per cent and 0.001 per cent a partial contraction of 
the pigment cells occurred in two minutes and thirty seconds. 
In a 0.0025 per cent curara solution the change took place in 
fourteen to forty minutes. In all the experiments the contrac- 
tion of the pigment cells was not evenly distributed but occurred 
in spots (fig. 15). The tail portion showed many contracted 
pigment cells, but in the head region there were the largest num- 
ber of contracted melanophores. Along the lateral line the pig- 
ment cells remained expanded. After fourteen or fifteen min- 
utes all the contracted pigment cells were expanded. This 
mixture of responses was constant for all the experiments. 



CHEMICAL AGENTS ON CHROMATOPHORES 177 

Pouchet ('76) observed that curara did not modify the reac- 
tion of the pigment cells of turbot, viz., the pigment cells re- 
mained in an expanded condition. Lode ('90) has found that 
subcutaneous injection of a mixture of curara and glycerine 
caused a dark coloration in the adult trout (Salmo fario). He 
ligated the aorta and found that in the anterior end with the 
intact circulation expansion of the pigment cells occurred, while 
in the posterior end with the interrupted circulation, the pig- 
ment cells remained contracted. These experiments are not 
conclusive because the removal of the circulation interfered 
with normal metabolism of the cells. Moreover, the pigment 
cells are very sensitive to the changes in their oxygen supply. 
He observed that if the spinal cord of a curarized trout was 
stimulated, no contraction of the pigment cells occurred. If the 
pigment cells were stimulated directly, the pigment cells con- 
tracted. He concluded that the curara destroyed the nerve 
endings but did not affect the pigment cells. Laurens ('15) 
found that if Amblystoma larvae were placed in a 0.2 per cent 
solution of curara their movements were abolished and the 
pigment cells remained expanded under all conditions. He con- 
cluded that this failure on the part of the pigment cells to react 
was probably due to the direct effect of the solution on the ani- 
mal; or asphjrxiation of the larvae by the curara, and the con- 
sequent increased amount of CO2 in the blood may have caused 
the melanophores to remain expanded. If a small amount of 1 
per cent solution of curara was injected into the body cavity the 
larvae were rendered unmotile but the melanophores reacted to 
light (expanded) and to darkness (contracted) as usual. He 
concluded here that this experiment did not prove that curara 
had no effect on the melanophores, for it has been shown that 
melanophores will contract and expand after all nervous con- 
nections have been destroyed. 

Carlson ('06) has shown that in weak solutions of curara there 
was a primary stimulation of the heart ganglion of Limulus. It 
had a httle effect on the heart muscle. Young ('81) observed 
that in Mya (sp.) and Solen (sp.) there was a distinct accelera- 
tion in the number of heart beats, and sometimes a diminution, 



178 JOHN N. LOWE 

and even a complete arrest. Plateau ('80) found that curara 
did not modify the frequency of the amplitude of the Decapod 
heart. Larger doses diminished the amplitude. Dogiel (77) 
showed there was a primary stimulation by curara of the heart 
of Corethra plumicornis. Boehm and Tillie ('04) have observed 
a primary stimulation of the isolated mammaUan heart (dog). 

Since Curara stimulates the central nervous system Cushny 
('10) and other ganglia Carlson ('06), it is possible that it acts 
as a stimulant on the medulla and spinal cord which transmit 
the impulse to the chromatophores, and a contraction results. 
Later as the curara destroys the nerve end plates the stimulus 
does not reach the pigment cell from the center. The pigment 
cells retain their independent irritability for a long time. The 
mixture of contracted and expanded melanophores is probably 
due to the unequal action of the curara on the peripheral nervous 
mechanism of the melanophores. 

6. Nicotine. When trout embryos were exposed to 0.5 per cent 
nicotine solution, their muscles twitched for a moment and then 
all activity ceased. The heart beat continued for twenty-eight 
minutes. There was no change in the pigment cells. The pig- 
ment cells disintegrated soon after death. There was a very 
marked maceration of all the tissues. The whole fish was cov- 
ered by a colorless slime. In a 0.125 per cent nicotine solution 
there was a slight primary contraction of the melanophores 
which was followed almost simultaneously by an expansion. 
The eyes bulged out of the head which caused the fish to ap- 
pear grotesque. A 0.005 per cent solution of nicotine caused a 
complete contraction of the pigment cells in two and one-half 
minutes. The paralytic expansion occurred eight minutes after 
the contraction. In 0.0025 per cent nicotine the contraction 
time was the same as in the preceding experiment. The period 
of paralysis was delayed which occurred in eleven minutes. A 
nicotine solution of 0.0005 per cent produced a complete con- 
traction in eleven minutes. The paralysis or depression of the 
pigment cells appeared in thu'ty-five minutes. In a 0.0001 per 
cent nicotine solution there occurred only a ^-ery slight change 
in the form of the pigment cells. In diluted 0.00005 per cent 



CHEMICAL AGENTS ON CHROMATOPHORES 179 

no change was elicited. In all the cases where paralytic expan- 
sion occurred, it appeared first on the ventral side. The reason 
for this is not understood. 

Redi (1634) according to van Rynberk ('05) observed that 
eels which died in a tobacco solution became lighter in color. 
Cushny ('10) says, that in nicotine the spinal cord is thrown 
into a condition of exaggerated irritability and that the medulla 
seems to be involved to a gi'eater degree than the spinal cord. 
The stimulation does not involve the higher brain centers. Carl- 
son ('06) observed that nicotine in weak solutions stimulated the 
heart ganglion of the Limulus heart. This primary stimulation 
was followed by a depression. There was no primary stimula- 
tion of the heart muscle. Gee ('13) has found that in a solution 
of 0.00066 per cent of nicotine leeches were vigorously stimu- 
lated, which was followed by a depression of movements. Ro- 
manes ('77) found that violent spasms were incited in the 
medusae Sareia (sp.) and Tiaropsis when exposed to nicotine. 
He also observed various distortions. Langley and Dickson 
('90) concluded that nicotine acts directly on the nerve cells and 
not on the muscle. 

It is known that nicotine first stimulates and later paralyzes 
the ganglionic cells of the sympathetic system, whether appUed 
directly to them or injected into the circulation. It is quite 
probable that nicotine affects the sjmipathetic system of the 
pigment cells, for there is first a contraction of the cells which is 
later followed by an expansion. 

7. Atropine. Strong solutions (0.5 per cent) produced no change 
in the pigment cells. The fish lived four hours in this concen- 
tration. In 0.025 per cent solution of atropine sulphate, there 
was no change in the pigment cells. All possible concentrations 
were tried, but none of them produced a contraction of the 
pigment cells. 

Pigment .cells were contracted in 0.005 per cent strychnine 
solution and then were transferred to solutions of 0.05 per cent 
to 0.0025 per cent of atropine, where all the pigment cells ex- 
panded rapidly. The expansion was complete from two to 
four minutes. Pigment cells contracted in potassium salts were 
expanded just as those that were contracted by strychnine. 



180 JOHN N. LOWE 

All the experiments show conclusively that atropine does not 
have any direct stimulating action on the pigment cells of trout 
embryos. Cushny ('10) says, that atropine acts on the higher 
centers of the brain and less on the lower divisions, viz., the 
medulla and the spinal cord, which is just the reverse of strych- 
nine. This acts on the lower centers and not on the central 
system. The results obtained justify the conclusion that the 
pigment cells are controlled by the lower reflex centers. 

Romanes ('77) in his experiments on the medusae Sarsia (sp.) 
and Tiaropsis found that atropine caused convulsive swimming 
movements by a marked depression. Pickering ('93) showed that 
0.012 gm. of atropine to 1 cc. of normal saline solution reduced 
the normal heart beat of the embryonic heart of the chick. 
Carlson ('06) found that atropine stimulated the heart ganglion 
and not the muscle of the Limulus heart. Cushny ('10) says most 
secretions are depressed by the administration of atropine. 
This is not due to the inactivation of the secretory cells, but to 
the failure of the nervous impulses. The action of atropine on 
other tissues, from all evidence, shows us that it does not act 
directly on the vascular and secretory elements, but on their 
nerve terminations. It is therefore possible that atropine acts 
on the pigment cells thi-ough their nerve fibers, paralyzing them, 
but does not act directly on the pigment cells. 

8. Cocaine. One-half per cent solutions killed the trout em- 
bryos rapidly. There was a momentary stimulation of the pig- 
ment cells which was followed almost simultaneously by an 
expansion of the pigment cells. In a 0.125 per cent solution of 
cocaine the behavior of the pigment cells was the same as in the 
0.5 per cent solution. In 0.025 per cent to 0.05 per cent cocaine 
solution the pigment cells were contracted in four minutes. The 
contraction was followed by an expansion. Solutions of 0.005 
per cent of cocaine produced a complete contraction in five 
minutes. The expansion followed in twelve minutes. Very 
weak, 0.00033 per cent solutions, had no effect on the melano- 
phores. 

These results show that cocaine has a primary stimulating 
action on the pigment cells of trout embryos. This primary 



CHEMICAL AGENTS ON CHROMATOPHORES 181 

stimulation is followed by an expansion of the pigment cells. 
The action of cocaine on the nervous system is in a series, 
namely, the cerebrum is first affected, then the cerebellum and 
medulla, and lastly the spinal cord. It also acts on the sen- 
sory fibers and theu- terminations. 

Von Frisch ('11) observed that the local application of cocaine 
caused the contraction of the melanophore in the minnow and 
Carssius (sp.). An injection of a 5 per cent solution into the body 
cavity caused a contraction of the pigment cells after the sym- 
pathetic nerves were severed. He concluded that the action 
of cocaine was through the central nervous system. Carlson 
('06) showed that weak solutions of cocaine had a primary stim- 
ulating action on the heart ganglion of Limulus, but had no 
effect on the heart muscle. Hedborn ('99) observed a slight 
primary stimulating action on the isolated heart of the cat. 

It is probable that cocaine acts on the reflex center which 
controls the pigment cells. It may act on the nerve endings of 
the pigment cells. It is obvious that it will require a great deal 
more of work to determine the relation of cocaine to the pigment 
cells before any generalization can be made. 

9. Veratrine. Solutions of veratrine of 0.5 per cent concen- 
tration caused a rapid contraction of the pigment cells. The 
contraction was complete in two minutes. Paralysis set in at 
six minutes, and pigment cells were completely expanded in 
two more minutes. In a 0.25 per cent solution the stages were 
the same. In a 0.005 per cent veratrine solution the pigment 
cells were completely contracted in nine minutes. The first 
signs of paralysis appeared in eighteen minutes. Veratrine 
was a very active agent in causing the contraction of the pig- 
ment cells. Dilutions of 0.0005 per cent to 0.00005 per cent 
caused a contraction of the pigment cells in eighteen to twenty 
minutes. The paralytic expansion occurred in thirty minutes. 
A solution of 0.00001 per cent veratrine caused no change in 
the pigment cells. 

Veratrine acts on the medullary center and the spinal cord, 
where a marked increase of irritability is elicited. After large 
doses there is a paralysis of the centers. It acts on the periph- 



182 JOHN N. LOWE 

eral ganglia and nerve endings. It is highly probable that the 
action of veratrine on the pigment cells is through the lower 
centers of the nervous system rather than local. 

Carlson ('06) found that weak solutions of veratrine had a 
primary stimulating action on the heart ganghon of Limulus. In 
strong solutions the period of stimulation was followed by a de- 
pression in two minutes. The ganglion free heart did not re- 
spond to the poison. Plateau C78) observed a primary stimu- 
lation in the heart of Carcinus moenas and Homarus (sp.) which 
was followed by a depression. Romanes ('77) found- that in the 
medusa Sarsia (sp.) the first effect of veratrine was an increase in 
the number and potency of the contractions. This period of 
increased responsiveness was followed by a gradual depression 
into complete quiescence. 

Summarizing the action of veratrine on the pigment cells, it 
may be stated, that it first stimulates the contraction of the 
pigment cells. This period of stimulation is followed later by a 
paralysis of the mechanism controlling the pigment cells. The 
pigment cells are expanded during this period of depression. 
This is in harmony with the observations of other workers on 
various tissues, where there is observed a primary stimulation 
followed by a depression. 

10. Quinine. Quinine in a 0.5 per cent maintained for a long 
time the pigment cells in an expanded condition. Dilutions were 
made from 0.25 per cent to 0.0005 per cent of quinine hydrochlo- 
ride solution, and in all of these dilutions no change occurred in 
the pigment cells. Solutions of 0.000033 per cent to 0.0000165 
per cent gave the same result. 

The pigment cells were first contracted in picrotoxin and were 
then placed in the quinine solutions of 0.025 per cent to 0.005 
per cent and in every case a rapid expansion of the pigment cells 
occurred. The rapidity of the expansion was greater in the 
quinine than it was in the ordinary process of washing out of 
the picrotoxin. 

Quinine differs from most drugs in that its action is very 
widespread, and it is often called a general protoplasmic poison. 
Binz ('68) observed that quinine inhibited the beat of the ciUa 



CHEMICAL AGENTS ON CHROMATOPHORES 183 

in protozoans. Also, that it stopped the movements of the 
leucocytes. Santesson ('93) found that quinine depressed the 
rhythm of an isolated frog's heart. Hedborn ('99) observed 
that quinine depressed an isolated mammalian heart (cat). 
0. and R. Hertwig ('87) observed that sperm treated with qui- 
nine had their movement paralyzed. Eggs when treated with 
quinine after the sperm entered, the conjugation of the pro- 
nuclei was delayed. Carlson ('06) has found that quinine did 
not stimulate the ganglion or the muscle of the Limulus heart. 
It is obvious from the experiments that quinine exhibits no 
primary stimulating action on the pigment cells of trout em- 
bryos. Any accurate interpretation of the depressing action of 
quinine is not possible, since the drug acts in the same way on 
the nervous tissues and the pigment cells as well. 

SUMMARY 

1. The experiments were performed on the melanophores 
(pigment cells) of the brook trout embryos, Salvehnus fontinalis 
Mitchill. Such young trout have only one kind of pigment 
cells, the melanophores. The young two-day or two-week old 
trout do not yet react to back ground. The fii-st sign of reac- 
tion to back ground appears only after the yolk is absorbed. 

2. In the presence of oxygen the pigment cells remain ex- 
panded and the fish Uve indefinitely. When hydrogen (oxygen 
want) is substituted for the oxygen the pigment cells contract 
and the embryos die. Oxygen is necessary for the maintenance 
of the expansion of the melanophores and life of the trout 
embryos. 

3. Carbon dioxide excess caused a contraction of the melano- 
phores. If oxygen was bubbled with the carbon dioxide, the 
presence of the oxygen had an antagonistic action. 

4. Distilled water caused a rapid contraction. A mixture of 
distilled and boiled tap water gave the same result. In boiled 
tap water the pigment cells contracted. Oxygenated distilled 
water and boiled tap water maintained the pigment cells in a 
normal expanded condition. It was the absence of oxygen and 
not of the salts that caused the contraction. 



184 JOHN N. LOWE 

5. In the potassium salts, K2SO4, KCl, KBr, KNO3, and KI 
there occurred a rapid contraction of the expanded melano- 
phores. The rate and degree of the contraction was the order 
given 

I> N0,> Br> Cl> SO4 

This primary contraction was followed by a cytolytic de- 
generation (expansion) . The time required for the appearance 
of this degeneration was greatest in 

I> N03> Br> Cl> SO4 

If the contraction or degeneration of the melanophores is 
specific for the potassium cation, it is unqualifiedly modified by 
its anion, or the residual part of the undissociated molecules. 

6. The neutral salts of sodium, Na2S04, NaCl, NaBr, NaNOs, 
and Nal, caused a slow contraction of the melanophores. The 
contraction was most rapid in Nal and slowest in Na2S04 and 
other salts were intermediate as 

I> N03> Br> Cl> SO4 

Degeneration appeared first in Nal and last, in Na2S04 and 
varied in this order 

I> N03> Br> Cl> SO4 

The irritabihty of the chromatophores and life of the fish was 
maintained longest in Na2S04 and NaCl from (118 to 132 hours) 
in Nal from one to two and one-half hours. 

7. The pigment cells that were contracted in potassium salts, 
when placed in sodium salt they expanded. The order of ex- 
pansion was 

S04> Cl> Br> N03> I 

There was no expansion in Nal. 

8. The results obtained in the experiments on the action of 
the salts on the pigment cells of trout are probable to be ex- 
plained on one or more of three assumptions: (1) That it is due 
to the antagonistic action between anion and cation ; (2) that it 
is the independent action of the cation; (3) that reaction of the 
melanophores is likely modified by the undissociated molecule. 



CHEMICAL AGENTS ON CHROMATOPHORES 185 

9. The narcosis or depression of the pigment cells of trout by 
the homologous alcohols corresponds very closely to their nar- 
cotic action as determined by Overton and numerous other 
investigators. 

10. Very dilute solutions of methyl, ethyl, and propyl alcohols 
exert no action on the pigment cells of trout. 

11. The pigment cells of trout embryos respond to alcohoUc 
stimuli. Their mode of reaction is comparable to .the reaction 
of other tissues to alcohols inasmuch as they are stimulated by 
small doses and depressed by large doses. 

12. Strychnine in moderate doses causes a primary con- 
traction of the expanded melanophores. Large doses cause a 
depression without a primary stimulation (contraction). The 
action of the strychnine is on the nervous system rather than 
on the pigment cells directly. 

13. The action of picrotoxin causes a rapid contraction of 
the pigment cells. The mechanism controlling the pigment 
cells is in the higher centers, because if the spinal cord is severed 
the pigment cells expanded. 

14. Morphine induces a contraction of the melanophores in 
isolated areas. This is probably due to the selective action of 
morphine upon the nervous system. Large doses produce no 
change in the expanded melanophores. Morphine expands the 
pigment cells that were contracted in picrotoxin, KCl, and 
strychnine. 

15. Curara causes a mixture of responses, that is, there are 
areas of expanded and contracted melanophores. This is likely 
due to the unequal action of the curara.on the peripheral nervous 
mechanism of the melanophores. 

16. Medium solutions of nicotine cause a contraction of the 
pigment cells. Strong nicotine solutions have no effect on the 
pigment cells. The action of nicotine is diz-ectly on the nervous 
controlling mechanism of the pigment cells. 

17. Atropine in all concentrations has no stimulating action 
on the pigment cells of trout. Atropine paralyzes the fine nerve 
connections of the pigment cells. 



I 



186 JOHN N. LOWK 

18. Cocaine has a primary stimulating action on tlie pigment 
cells of trout. This action is probably on the nerve endings of 
the pigment cells that connect them with the reflex center. 

19. Veratrine causes a primary contraction of the pigment 
cells which is followed by a rapid depression (expansion). The 
action of veratrine is through the reflex center of the spinal 
cord and medulla rather than local. 

20. Quinine exhibits no primary stimulating action on the 
pigment cells. The drug has no selective action on tissues, 
therefore it is a general 'protoplasmic poison.' 



CHEMICAL, AGENTS ON CHROMATOPHORES 187 

BIBLIOGRAPHY 

Baer, G. 1S9S Beitrag zur Kenntniss der acuten Vergiftung mit verschiedenen 

Alkoholen. Arch. f. (Anat. u.) Phys., Leipz., S. 283-296. 
Ballowitz, E. 1893 Die Nervenendigungen der Pigmentzellen, ein Beitrag 

zur Kenntnis des Zusammemlianges der Endverzweigungen der Ner- 

venmit dem Protoplasma der Zellen. Zeitsclir. f. wiss. Zool., Bd. 56, 

S. 673-706. 
Benedict, Stanley R. 1905 The role of certain ions in rhythmic heart ac- 
tivity. Amer. Jour, of Phys., vol. 8, pp. 192-204. 

1908 The influence of salts and non-electrolytes upon the heart. 

Amer. Jour, of Phys., vol. 22, pp. 16-31. 
BiNZ, C. 1873 Ueber Chinin und Blut., Arch. f. exp. Path. u. Pharm., Bd. 1, 

S. 18-30. 

1876 Literarische Notizen zum vorstehenden Thema. Arch. f. exp. 

Path. u. Pharm., Bd. 5, S. 39-.54. 

1878 Zur Salicylsaure- und Chininwirkung., Arch. f. exp. Path. u. 

Pharm., Bd. 7, S. 275-316. 
Bradbury, J. B. 1899 Some points connected with sleep, sleeplessness and 

hypnotics. Brit. Med. Jour., vol. 2, pp. 4-9. 
Carlson, A. J. 1906 On the point of action of drugs on the heart with special 

reference on the heart of Limulus. Amer. Jour. Phys., vol. 17, p. 

177-210. 
Cololian, p. 1901 La Toxicity des Alcohols chez les Poissons. Jour, de 

Phys. et de Path., T. 3, pp. 535-546. 
CusHNY', A. R. 1910 Pharmacology and Therapeutics or the action of Drugs. 

(Lea Bros. & Co., Philadelphia), pp. 744. 
DoGiEL, J. 1877 Anatomie und Physiologic des Herzens der Larvae von 

Corethra plumicornis. Memor. de L'Acad. Imper. des Sciences de 

St. Petersburg. T. 24, pp. 1-37. 
v. Frisch, Karl 1911 Beitriige zur Physiologic der Pigmentzellen in der 

Fischhaut. Pfltiger's Arch. f. d. ges. phys., Bd. 138, S. 319-388. 
FncHS, R. F. 1914 Der Farbenwechsel und die chromatische Hautfunktion 

der Tiere. Handbuch der vergleichenden Physiologic, Bd. 3, S. 

1189-1656. 
Gee, Wilson 1913 The behavior of leeches with especial reference to its 

modifiability. Univ. of Cal. pub. (In Zoology), vol. 11, pp. 197- 

305. 
Hamburger, H. J. 1891 Ueber den Einfluss der Athmung auf die Permeabili- 

tat der Blutkorperchen. Zeitschr. f. Biol., Bd. 28, S. 405-416. 
Hamburger, H. J. und van Leir, G. Ad 1902 Die Druchlassigkeit der rothen 

Blutkorperchen fiir die Anion von Natrium. Arch. f. Anat. u. Phys., 

Bd. S. 492-532. 
Hedborn, Karl 1899 Ueber Einwirkung verschiedener Stoffe auf das isolirte 

Saugethierherz. Skand. Arch. f. Phys., Bd. 9, S. 1-72. 
Hertwig, O. und Richard 1887 Ueber den Befruchtungs- und Teihmgsvor- 

gang des tierischen Eies unter dem Einfluss jiusserer Agentien. Jena- 

ische Zeit. f. Med. und Naturwiss., Bd. 20, S. 120-241 und 477-510. 



188 JOHN N. LOWE 

Howell, W. H. 1S9S On the relation of the blood to the automaticity and 
sequence of the heart-beat. Amer. Jour, of Phys., vol. 2, pp. 47-81. 

JoPFROY, A. ET Severeatix 1895 Nouveau Precede de Mensuration de la 
Toxicito des Liquides, parla Methode das Injections Intra-Veneuses 
Application a la Determination de la Toxicite des Alcools. Archives 
De Medicine Experimentale, 1895, pp. 569-588. 

Kahlenberg, L. and True, R. H. 1896 On the toxic action of dissolved 
salts and their electrolytic dissociation. Bot. Gaz., vol. 22, pp. 81- 
124. 

KoBERT, E. R. 1882 Ueber den Einfluss verschiedener pharmakologischer 
Agentien auf die Muskelsubstanz. Arch. f. exp. Path. u. Pharm., Bd. 
15, S. 22-80. 

1893 Ueber die Wirkung einiger China- Alkaloide auf das isolirte 
Froschherz und auf den Blutdruck des Kaninchens. Arch. f. exp. 
Path. u. Pharm., Bd. 32, S. 321-371. 

Koch, W. 1909 Die Bedeutung der phosphatide (Lecilhane) fiir die lebende 
Zelle. II. Zeitschr. f. physiol. Chem., Bd. 63, S. 432-442. 

Langley, J. N. AND Dickson, VV. L. 1890 Pituri and Nicotin. Jour. Phys. 
Vol. II, pp. 265-306. 

Laurens, H. 1915 The reactions of the melanophores of Amblystoma larvae. 
Jour. Exp. Zool., vol. 18, pp. 577-6.38. 

Lee, F. S. 1902 The action of ethyl-alcohol on contractile protoplasm. Amer. 
Jour, of Phys., vol. 8, p. 19. 

Lee, F. S. and Salant, W. 1902 The action of alcohol on muscle. Amer. 
Joiir. Phys., vol. 8, pp. 61-74. 

LiLLiE, R. S. 1911 Certain means by which starfish eggs naturally resistant 
to fertilization may be rendered normal and the physiological con- 
ditions of this action. Biol. Bull., vol. 22, pp. 328-346. 

1911 Antagonism between salts and anaesthetics. I. On the con- 
ditions of the anti-stimulating action of anaesthetics with observa- 
tions on their protective or antitoxic action. Amer. Jour. Phys., vol. 
29, pp. 372-397. 

1912 Antagonism between salts and anaesthetics. II. Decrease by 
anaesthetic in the rate of toxic action of pure isotonic salt solutions 
on unfertilized starfish and sea urchin eggs. Amer. Jour. Phys., 
vol. 30, pp. 1-17. 

LiNGLE, D. J. 1900 The action of certain ions on ventricular muscle. Amer. 

Jour, of Phys., vol. 4, pp. 265-282. 
Lode, Alois. 1890 Beitrage zur Anatomie und Physiologic des Farbenwech- 

sels der Fische. Sitz. d. kaiserl. Akad. d. wiss. zu Wien. Math natur- 

wiss. Kl., Bd. 99, Abt. 3, S. 130-143. 
LoEB, J. 1900 Ueber die Bedeutung der Ca- und K-ionen fiir die Hertzthatig- 

keit. Pfliiger's Arch. f. d. ges. Phys., Bd. 88, S. 229-232. 

1900 On ion-protein compounds and their role in the mechanics of 

life phenomena. I. The poisonous character of a pure NaCl solution. 

Amer. Jour, of Phys., vol. 3, pp. 327-338. 

1900 On the different effect of ions upon myogenic and neurogenic 

contractions and upon embryonic and muscular tissue. Amer. Jour. 

of Phys., vol. 3, pp. 383-396. 



CHEMICAL AGENTS ON CHROMATOPHORES 189 

LoEB, J. 1902 Ueber den Einfluss der Werthigkeit und moglioher antitoxische 
Wirkung. Pflliger's Arch. f. d. ges. Phys., Bd. 88, S. 68-78. 

1910 Further experiments on the antagonistic action of salts. Amer. 
Jour, of Phys., vol. 27, pp. 22-23. 

LoEB, J. UND Wastenbys, H. 1911 a Die Entgiftung von Kaliumsalzen durch 
Natriumsalze. Biochem. Zeitschr., Bd. 31, S. 450-477. 

1911 b Die Erhohung der Giftwirkung von KCl durch niedrige Kon- 
zentration von NaCI. Biochem. Ztschr., B-31, S. 155-163. 

1912 Abhangigkeit der relativcn Giftigkeit von Na und Ca von 
Anion. Biochem. Zeitschr., Bd. 39, S. 194-199. 

LoEB, J. UND Cattell, McK. 1915 The influence of electrolytes upon the dif- 
fusion of potassium out of the cell and into the cell. Jour, of Biolog. 
Chem., vol. 23, pp. 41-66. 

LoEB, O. 1905 Die Wirkung des Alkohols auf Warmbluterherz. Arch. f. exp. 
Path. u. Pharm., Bd. 52, S. 459-480. 

M.^THEws, A. P. 1902 The nature of nerve stimulation and of changes in irri- 
tability. Science, vol. 15, pp. 492-498. 

1904 a The relation between solution tension atomic vol. and 
the physiological action of the elements. Amer. Jour, of Phys., vol. 
10, pp.' 290-323. 

1904 b The cause of the pharmacological action of the iodates, bro- 
mates, chforates, other oxidizing substances and some organic drugs. 
Amer, Jour, of Phys., vol. 11, p. 237-249. 

1904 c The nature of chemical and electrical stimulation. 

I. The physiological action of an ion depends upon its electrical state 
and its electrical stability. Amer. Jour, of Phys., vol. 11, pp. 455-496. 

1905 The nature of chemical and electrical stimulation. II. The ten- 
sion coefficient of salts and the precipitation of colloids by electro- 
lytes. Amer. Jour, of Phys., vol. 14, pp. 203-230. 

1905 The toxic and antitoxic action of salts. Amer. Jour, of Phys., 
vol. 12, pp. 419-443. 

1906 A contribution to the general principles of the pharmodynamics 
of salts and drugs. Biological Studies by the Pupils of William 
Thompson SedgH-ick, pp. 81-118. 

McClendon, J. F. 1910 How could increase in permeability to electrolytes 

allow the development of the egg? Proc. of the Soc. for Exp. Biol. 

and Med., vol. 8, pp. 1-3. 
Meyer, Hans 1899 Zur Theorie der Alkoholnarkose. Arch. f. exp. Path, und 

Pharm., Bd. 42, S. 109-137. 
Overton, E. 1901 Studien tib'er die Narkose zugleich ein Beitrag zur AUge- 

meinen Pharmakologie. (Verlag von Gustav Fischer), S. 195. 
Paul, T. und Kronig, B. 1896 Ueber das Verhalten der Bakterien zu chemis- 

chen Regaentien. Zeitschr. f. Physikal. Chem., Bd. 21, S. 414-450. 
Picaud, M. 1897 Sur la toxicite des alcools. Comptes Rendus, T. 124, pp. 

829-830. 
PiCKERiNO, J. W. 1893 Observation on the physiology of the embryonic heart. 

Jour. Phys., vol. 14, pp. 383-466. 

THE JOURNAL OF EXPERIMENT.\L ZOOLOGY, VOL. 23, NO I 



190 JOHN N. LOWE 

Pickering, J. W. 1895 Further experiments on the embryonic heart. Jour. 

of Phys., vol. 18, pp. 470-483. 
PoucHET, G. 1872 Du role des nerfs dan les changements de coloration des 

poissons. Jour, de L'Anat. et de Phys., T. 8, pp. 71-74. 

1876 De changements de coloration sous I'influence des nerfs. Jour. 

de I'Anat. et de Phys., vol. 12, pp. 1-90, 113-165. 
Plate.^u, Felix 1880 Recherches Physiologiques sur le Coeur des Crustac^s 

Decapodes. Arch, de Biol., T. 1, pp. 595-695. 
Redi, F. 1664 Osservazioni intorno alle Vipre. Firenze 4. 
Romanes, G. J. 1877 Further observations on locomotor system of Medusae. 

Trans. Roy. Soc. London, vol. 167, pt. II, pp. 659-752. 

1885 Jelly-fish, Star-fish, and Sea Urchins. Internat'l Scien. Series 

(D. Appleton & Co.), pp. 323. 
VAN Rynberk, G. Ueber den durch Chromatophoren bedingten. Farben- 

wechsel der Tiere (sog. Chromatische Hautfunktion). Ergebnisse 

der Phys., Bd. 5, S. 347-571. 
Santessen, C. G. 1892 Ueber den Einfluss einiger China Alkaloide auf die 

Leitstungsfiihigkeit der Kaltbliitermuskeln. Arch. f. exp. Path. u. 

Pharm., Bd. 30, S. 411-447. 
Scheffer, J. C. Th. 1900 Studien uber den Einfluss des Alkohols auf die 

Muskelarbeit. Arch. f. exp. Path. u. Pharm., Bd. 44, S. 24-58. 
SoLLOMANN, T. 1906 Text-Book of Pharmacology. (W-. B. Saunders Co.), 

pp. 1070. 
Spaeth, R. A. 1913 The phj'siology of the chromatophores of fishes. Jour. 

Exp. Zool., vol. 15, pp. 527-579. 

1916 Evidence proving the melanophore to be a disguised type of 

smooth muscle cell. Jour. Exp. Zool., vol. 20, pp. 193-213. 
Vernon, H. M. 1910 The mode of union of certain poisons with cardiac 

muscle. Jour, of Phys., vol. 41, pp. 194-232. 

1911 The action of homologous alcohols and aldehydes on the tor- 
toise heart. Jour, of Phys., vol. 43, pp. 325-342. 
Waller, A. D. 1896 On the influence of reagents on the electrical excita- 
bility of isolated nerve. Brain, vol. 19, pp. 43-67, 277-300. 

1908 The comparative effect upon striped muscle of alcohol, ether, 
and chloroform. Jour, of Phys., vol. 37, pp. 77-94. 

1909 The comparative power of alcohol, ether and chloroform as 
measured by their action upon isolated muscle. Proc. Roy. Soc, 
vol. 81, pp. 545-558. 

WiRGiN, G. 1904 Vergleichende Untersuchung fiber die keimtodtenden und 
entwickelungshemmenden Wirkungeti von Alkoholen der Methyl-, 
Aethyl-, Propyl-, Butyl-, und Amylreihen. Zeitsch. f. Hyg., Bd. 44, 
S. 149-168. 

Wood, H. C. and Hoyt, H. D. 1905 The action of alcohol upon the circulation. 
National Acad, of Science, (Memoirs), vol. 10, pp. 43-70. 

Yung, Emile 1881 De I'innervation du Coeur et de I'Action des Poisons chez 
les MoUusques Lamellebranches. Arch, de Zool. Exp., T. 9, pp. 
429-144. 



CHEMICAL AGENTS ON CHROMATOPHORES 191 

Ytjng, Emile 1882 Recherches experimentales sur I'action des poisons chez les 
c^phalopodes. Mitteilungen aus d. Zool., S tat. zu Neapel., Bd 3 
pp. 97-120. 

ZoETHOUT, W. D. 1904 The effects of various salts on the toxicity of skeletal 
muscles. Amer. Jour, of Phys., vol. 10, pp. 211-221. 



PLATE 1 

• EXPLANATION OP FIGURES 

In figures 1 to 10 are shown five sets of brook trout embryos which were ex- 
posed to the action to 0.2 \l solutions of Kl, KNO3, KBr, KCl, and K2SO4, 1 to 
5 for an interval of fifteen minutes, and figures 6 to 10 for a period of three 
hours. 

1 The melanophores were completely contracted in Kl. 

2 The contraction was not as pronounced in KNO3. 

3 In KBr the melanophores had longer processes than in the two preceding 
solutions. 

4 In KCl the processes were more distinct and showed the finer arboriza- 
tions. 

5 An exposure of fifteen minutes to K2SO4 produced no observable change 
in the melanophores. 

6. After an exposure of three hours to Kl the melanophores showed a distinct 
secondary expansion. 

7 In KNO3 an exposure of three hours produced a less extensive secondary 
expansion than Kl. 

8 In KBr the processes were very much shorter than in- Kl and KNO3. 

9 After three hours in KCl the melanophores were still spherical, but there 
was a suggestion toward a peripheral migration of the pigment as indicated by 
the swollen condition of the cells. 

10 After three 'hours in KjSOj there was no expansion of the melanophores. 

11 All melanophores contracted, photograph taken after twenty-four hours 
of exposure to Picrotoxin. 

12 The melanophores expanded after severing the tail in an individual which 
was exposed to Picrotoxin for twenty-four hours. Photograph taken five min- 
utes after cutting. 

13 All melanophores contracted during the period of Strychnine con- 
vulsions. 

14 Showing the expansion of the melanophores after the strychnine convul- 
sion had subsided. 

15 The contracted and expanded melanophores as they occurred in 0.002.5 
per cent curara 



CHEMICAL AGENTS ON C 

JOHN N. LO 


HROMATOPHORES 

It * ^ 




PLATE 1 

• • 












• 4 



•■|:3 




? 



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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 

VOLUME 23, NUMBER 1, MAY, 1917 



CONTENTS 

Harley N. Gould. Studies on sex in the hermaphrodite muUusc Crepidula plana. I. 
History of the sexual cycle. Eighty-five figures 1 

Henry Laurens and J. W. Williams. Photomechanical changes in the retina of normal 
and transplanted eyes of Amblystoma larvae. Three text figures and one plate. ... 71 

Franklin Pearce Reagan. The role of the auditory sensory epithelium in the forma- 
tion of the stapedial plate. Ten figures 85 

Edwin Carleton MacDowell. Bristle inheritance in Drosophila. II. Selection. 
Ten figures 109 

John N. Lowe. The action of various pharmacological and other chemical agents on 
the chromatophores of the brook trout Salvelinus fontinalis Mitchill. Three text 
figures and one plate 147 

IIenrt Laurens. The reactions of the melanophores of Amblystoma tigrinum larvae to 
light and darkness. Six figures 1P5 

Caret Pratt McCord and Flotd P. Allen. Evidences associating pineal gland function 
with alterations in pigmentation. Seven figures 207 



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